Structure determination of five sialylated trisaccharides with core types 1, 3 or 5 isolated from bovine submaxillary mucin

In this study we have investigated the structures of five sialylated trisaccharides released from bovine submaxillary mucin by alkaline borohydride treatment and isolated by high-performance liquid chromatography. Three of the trisaccharides contained NeuAc while two contained NeuGc. One oligosaccharide contained core-type 1, two contained core-type 3 and two contained core-type 5. The structures, determined by a combination of one- and two-dimensional 1H-NMR spectroscopy at 270 MHz and methylation analysis involving gas-liquid chromatography/mass spectrometry, were as follows: A4b, GalNAc alpha(1----3) [NeuAc alpha(2----6)]GalNAcol; A4c, GlcNAc beta(1----3)[NeuAc alpha(2----6)]GalNAcol; A4d, Gal beta(1----3)[NeuAc alpha(2----6)]GalNAcol; A4e, GalNAc alpha(1----3)-[NeuGc alpha(2----6)]GalNAcol; A4f, GlcNAc beta(1----3)[NeuGc alpha (2----6)]GalNAcol. The oligosaccharides occurred in the approximate molar ratios 1.0:12.0:0.3:0.2:2.0. This is the first report of oligosaccharides containing core-type 5 and of the occurrence of oligosaccharides A4b, A4e, and A4f in bovine submaxillary mucin. 1H-NMR data for structure A4e, which is a novel structure, are presented for the first time.     

Structures of the sugar chains of mouse immunoglobulin G

The asparagine-linked sugar chains of mouse immunoglobulin G (IgG) were quantitatively liberated as radioactive oligosaccharides from the polypeptide portions by hydrazinolysis followed by N-acetylation, and NaB3H4 reduction. After fractionation by paper electrophoresis, lectin (RCA120) affinity high-performance liquid chromatography, and gel filtration, their structures were studied by sequential exoglycosidase digestion in combination with methylation analysis. Mouse IgG was shown to contain the biantennary complex type sugar chains. Eight neutral oligosaccharide structures, viz, +/- Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(+/- Gal beta 1---- 4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc, were found after the sialidase treatment. The molar ratio of the sugar chains with 2,1, and 0 galactose residues was 2:5:3. The galactose residue in the monogalactosylated sugar chains was distributed on Man alpha 1----3 and Man alpha 1----6 sides in the ratio of 1:3. The oligosaccharides were almost wholly fucosylated and contained no bisecting N-acetylglucosamine which is present in human, rabbit, and bovine IgGs.     

Mucin biosynthesis. Properties of a bovine tracheal mucin beta-6-N-acetylglucosaminyltransferase.

We have characterized a bovine tracheal mucin beta-6-N-acetylglucosaminyltransferase that catalyses the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the C-6 of the N-acetylgalactosamine residue of galactosyl-beta 1----3-N-acetylgalactosamine. Optimal enzyme activity was obtained between pH 7.5-8.5, at 5mM-MnCl2, and at 0.06-0.08% (v/v) Triton X-100 (or Nonidet P-40), or 0.5-5.0% (v/v) Tween 20. Ba2+, Mg2+ and Ca2+ could partially replace Mn2+, but Co2+, Fe2+, Cd2+ and Zn2+ could not. Sodium dodecyl sulphate, cetylpyridinium chloride, sodium deoxycholate, octyl beta-D-glucoside, digitonin and alkyl alcohols were less effective in enhancing enzyme activity, and dimethyl sulphoxide was ineffective. The apparent Michaelis constants were 1.25 mM for UDP-N-acetylglucosamine, 0.94-3.34 mM for freezing-point-depressing glycoprotein and 0.19 mM for periodate-treated blood-group-A porcine submaxillary mucin. Asialo ovine submaxillary mucin could not serve as the glycosyl acceptor. The structure of the 14C-labelled oligosaccharide obtained by alkaline-borohydride treatment of the product was identified as Gal beta 1----3(Glc-NAc beta 1----6)N-acetylgalactosaminitol by beta-hexosaminidase treatment, gas chromatography-mass spectrometry and 1H-n.m.r. (270 MHz) analysis. The enzyme is important in the regulation of mucin oligosaccharide biosynthesis.     

Structural studies on a binding site for Dolichos biflorus agglutinin in the small intestine of the mouse

Glycoproteins which bound to Dolichos biflorus agglutinin (DBA) were isolated from the small intestine of 129/Sv mice. Among oligosaccharides released from the carbohydrate moieties of the glycoproteins by endo-beta-galactosidase, the major one with N-acetylgalactosamine at the non-reducing end was isolated by QAE-Sephadex A-25 column chromatography. The structure of the oligosaccharide was elucidated to be GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4GlcNAc beta 1----3Gal by compositional analysis, methylation analysis before and after mild acid hydrolysis, sequential glycosidase digestion, secondary ion mass spectrometry (SIMS), and nuclear magnetic resonance spectroscopy. The SIMS signal of m/z 1,071 was consistent with the presence of the branched sequence, GalNAc(NeuAc)GalGlcNAc, and the signal was also detected in the high-molecular-weight fraction obtained after endo-beta-galactosidase digestion. The pentasaccharide identified here has the terminal structure of ganglioside GM2, and an apparently identical one has been identified as the epitope of blood group Sda and the DBA binding site in human T-H urinary glycoprotein. Thus, the present result has extended our knowledge of the biological meaning of the oligosaccharide structure and has established that GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4GlcNAc is a DBA binding site in the small intestine of the mouse.     

Binding specificity of a baby hamster kidney lectin for H type I and II chains, polylactosamine glycans, and appropriately glycosylated forms of laminin and fibronectin.

The carbohydrate binding specificity of Mr = 30,000 lectin (CBP30) from baby hamster kidney (BHK) cells has been studied by inhibition of binding of the radiolabeled lectin to asialofetuin-Sepharose using model oligosaccharides and glycopeptides. CBP30 binds type I or II Gal beta(1----3(4))GlcNAc chains but not Gal(beta 1----3)GalNAc. The inhibitory potency of straight chain polylactosamine structures or complex-type branched glycans is increased in proportion to the number of Gal(beta 1----3(4)) units present. Fucosylation or sialylation of terminal galactose residues or further substitution by (alpha 1----3)-linked galactose or N-acetylgalactosamine does not affect binding whereas substitution of the penultimate N-acetylglucosamine residue drastically reduces binding. Thus, blood group A, H type I or H type II structures, shows high affinity whereas Lex, Lea, and Leb structures bind poorly. CBP30 binds to murine Engelbreth-Holm-Swarm (EHS) tumor laminin and human amniotic fluid fibronectin but not human plasma fibronectin. Binding involves polylactosamine glycans as well as tri- and tetraantennary complex-type glycans present in EHS laminin and amniotic fluid fibronectin but absent in plasma fibronectin. Proteolytic fragments of EHS laminin (E1X/Nd, P1, E8, and E3) bind CBP30, but only fragment E8 supports attachment and spreading of BHK cells. BHK cell adhesion to EHS laminin or fragment E8 was not disturbed by CBP30-specific antibodies, but at relatively high concentrations (45 micrograms/ml) CBP30 inhibited spreading and partially attachment of cells on laminin.     

The structures of N- and O-glycosidic carbohydrate chains of a chondroitin sulfate proteoglycan isolated from the media of the human aorta

A large Mr chondroitin sulfate proteoglycan was extracted from the media of human aorta under dissociative conditions and purified by density-gradient centrifugation, ion-exchange chromatography, and gel filtration chromatography. Removal of a contaminating dermatan sulfate proteoglycan was accomplished by reduction, alkylation and rechromatography on the gel filtration column. After chondroitinase ABC treatment, the proteoglycan core was separated from a residual heparan sulfate proteoglycan by a third gel filtration chromatography step. As assessed by radioimmunoassay, the isolated proteoglycan core was free of link protein, but possessed epitopes that were recognized by antisera against the hyaluronic acid binding region of bovine cartilage proteoglycan as well as those that were weakly recognized by anti-keratan sulfate antisera. Following beta-elimination of the protein core, the liberated low Mr oligosaccharides were partially resolved by Sephadex G-50 chromatography, and their primary structure was determined by 500-MHz1H NMR spectroscopy in combination with compositional sugar analysis. The N-glycosidic carbohydrate chains, which were obtained as glycopeptides, were all biantennary glycans containing NeuAc and Fuc; microheterogeneity in the NeuAc----Gal linkage was detected in one of the branches. The N-glycosidic glycans have the following overall structure: (Formula: see text). The majority of the O-glycosidic carbohydrate chains bound to the protein core were found to be of the mucin type. They were obtained as glycopeptides and oligosaccharide alditols, and possessed the following structures: NeuAc alpha(2----3)Gal beta(1----3)GalNAc-ol, [NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----6)]GalNAc-ol, and NeuAc alpha-(2----3) Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)] GalNAc-ol. The remainder of the O-glycosidic carbohydrate chains bound to the isolated proteoglycan were the hexasaccharide link regions of the chondroitin sulfate chains that remained after chondroitinase ABC treatment of the native molecule. These latter glycans, which were obtained as oligosaccharide alditols, had the following structure (with GalNAc free of sulfate or containing sulfate bound at either C-4 or C-6): delta 4,5GlcUA beta(1----3)GalNAc beta(1----4)GlcUA beta(1----3)Gal beta(1----3)Gal beta(1----4)Xyl-ol.     

Structure of phosphonoglycosphingolipid containing pyruvylated galactose in nerve fibers of Aplysia kurodai

A phosphonoglycosphingolipid, designated as FGL-IIb, was identified in nerve fibers of Aplysia kurodai by two-dimensional thin layer chromatography (Abe, S., Araki, S., and Satake, M. (1986) Biomed. Res. (Tokyo) 7, 47-51). FGL-IIb was isolated from the nervous system of A. kurodai by Iatrobeads column chromatography using three solvent systems. Pyruvic acid was identified by thin layer chromatography as its 2,4-dinitrophenylhydrazone and established by permethylation studies to be attached as a ketal to O-3 and O-4 of the terminal galactose of the oligosaccharide chain in FGL-IIb. By sugar analysis, permethylation studies, fast atom bombardment-mass spectrometry, and proton magnetic resonance spectrometry, the structure of FGL-IIb was concluded to be [3,4-O-(1-carboxyethylidene)]Gal beta 1----3GalNAc alpha 1----3(Fuc alpha 1----2) (2-aminoethylphosphonyl----6)Gal beta 1----4Glc beta 1----1ceramide. Its major aliphatic components were palmitic acid, octadeca-4-sphingenine and anteisononadeca-4-sphingenine. This is the first report of the occurrence of pyruvylated galactose as a constituent of animal sphingolipid.     

Carbohydrate moieties of peanut agglutinin receptors isolated from human gastric cancer cells

Receptors for peanut agglutinin (PNA) were isolated from Kato III human gastric cancer cells by affinity chromatography on PNA agarose, and were labeled by the galactose oxidase-NaB3H4 method. Alkaline NaBH4 treatment of the labeled receptors released two small oligosaccharide alcohols, which were identified as Gal beta 1----3GalNAc-ol and Gal beta 1----4GlcNAc beta 1----6(Gal beta 1----3)GalNAc-ol. Higher oligosaccharides and glycopeptides of both N- and O-linked type were also detected, but they did not appear to bear PNA binding sites. The presence of oligo-N-acetyllactosamine units in the N-linked type sugars was indicated by endo-beta-galactosidase digestion.     

Characterization of a diphosphonopentaosylceramide containing 3-O-methylgalactose from the skin of Aplysia kurodai (sea hare)

The complete structure is proposed for a ceramide (Cer), bis(2-aminoethylphosphono)-pentaoside, isolated from the skin of Aplysia kurodai. This new phosphonoglycosphingolipid was purified using two systems of column chromatography on silicic acid. The purity of the glycolipid was confirmed by thin-layer chromatography, analysis of its composition, and proton magnetic resonance spectrometry. The component carbohydrates were glucose, galactose, N-acetylgalactosamine, and 3-O-methylgalactose. Most (90%) of the fatty acid was palmitic acid and the major sphingosine bases were octadeca-4-sphingenine (51%) and anteisononadeca-4-sphingenine (38%). 2-Aminoethylphosphonyl-6-galactose was identified after its partial hydrolysis. From studies by methanolysis, permethylation, mild acid hydrolysis, hydrogen fluoride treatment, chromium trioxide oxidation combined with thin-layer chromatography, gas liquid chromatography, gas chromatography-mass spectrometry, and proton magnetic resonance spectrometry, the structure of the glycolipid was concluded to be 3-OMeGal beta 1----3GalNAc alpha 1----3[6'-O-(2-aminoethylphosphonyl)-Gal alpha 1----2](2-aminoethylphosphonyl----6)Gal beta 1----4Glc beta 1----1Cer.     

Complete structure of the cell surface polysaccharide of Streptococcus oralis C104: a 600-MHz NMR study

Specific lectin-carbohydrate interactions between certain oral streptococci and actinomyces contribute to the microbial colonization of teeth. The receptor molecules of Streptococcus oralis, 34, ATCC 10557, and Streptococcus mitis J22 for the galactose and N-acetylgalactosamine reactive fimbrial lectins of Actinomyces viscosus and Actinomyces naeslundii are antigenically distinct polysaccharides, each formed by a different phosphodiester-linked oligosaccharide repeating unit. These streptococci all coaggregated strongly with both A. viscosus and A. naesludii strains, whereas S. oralis C104 interacted preferentially with certain strains of the latter species. Receptor polysaccharide was isolated from S. oralis C104 cells and was shown to contain galactose, N-acetylgalactosamine, ribitol, and phosphate with molar ratios of 4:1:1:1. The 1H NMR spectrum of the polysaccharide shows that it contains a repeating structure. The individual sugars in the repeating unit were identified by 1H coupling constants observed in E-COSY and DQF-COSY spectra. NMR methods included complete resonance assignments (1H and 13C) by various homonuclear and heteronuclear correlation experiments that utilize scalar couplings. Sequence and linkage assignments were obtained from the heteronuclear multiple-bond correlation (HMBC) spectrum. This analysis shows that the receptor polysaccharide of S. oralis C104 is a ribitol teichoic acid polymer composed of a linear hexasaccharide repeating unit containing two residues each of galactopyranose and galactofuranose and a residue each of GalNAc and ribitol joined end to end by phosphodiester linkages with the following structure. [----6)Galf(beta 1----3)Galp(beta 1----6)Galf(beta 1----6)GalpNAc(beta 1----3) Galp(alpha 1----1)ribitol(5----PO4-]n     

The carbohydrate moiety of band-3 glycoprotein of human erythrocyte membranes.

Band-3 glycoprotein was purified from human blood-group-A erythrocyte membranes by selective solubilization and gel chromatography on Sepharose 6B in the presence of sodium dodecyl sulphate. The purified glycoprotein was subjected to hydrazinolysis in order to release the carbohydrate moiety. The released oligosaccharides were N-acetylated and applied to a column of DEAE-cellulose. Most of the band-3 oligosaccharides obtained were found to be free of sialic acids. When this neutral fraction was subjected to gel chromatography on a column of Sephadex G-50, two broad peaks were observed indicating that the band-3 glycoprotein was heterogeneous in the size of the oligosaccharide moieties. All fractions from gel chromatography were found to contain galactose, mannose, N-acetylglucosamine and fucose. The higher-molecular-weight (mol.wt. 3000-8000) peak consisted of fucose, mannose, galactose, N-acetylglucosamine and N-acetylgalactosamine in a molar proportion of 1.6:3.0:8.4:10.5:0.2. Most of these oligosaccharides were digested with a mixture of beta-galactosidase and beta-N-acetylhexosaminidase after alpha-L-fucosidase treatment to give a small oligosaccharide with the structure alpha Man2-beta Man-beta GlcNAc-GlcNAc. Methylation studies and limited degradation by nitrous acid deamination showed that the oligosaccharides contained the repeating disaccharide Gal beta 1----4GlcNAc beta 1----3, with branching points at C-6 of some of the galactose residues. These results indicate that a major portion of the band-3 oligosaccharide has a common core structure, with heterogeneity in the numbers of the repeating disaccharides, and contains fucose residues both in the peripheral portion and in the core portion. Haemagglutination tests were also carried out to determine the blood-group specificities of the glycoprotein and the results demonstrated the presence of both blood-group-H and I antigenic activities.     

Structures of asparagine-linked oligosaccharides of human placental fibronectin

The asparagine-linked sugar chains of fibronectin purified from human placenta were quantitatively released as oligosaccharides by hydrazinolysis. After N-acetylation, they were converted to radioactive oligosaccharides by NaB3H4 reduction. The radioactive oligosaccharides were fractionated by their charge on an anion-exchange column chromatography. All of the acidic oligosaccharides could be converted to neutral oligosaccharides by sialidase digestion. These oligosaccharides were then fractionated by serial affinity chromatography using immobilized lectin columns. Study of each oligosaccharide by sequential exoglycosidase digestion and methylation analysis revealed the following information as to the structures of the sugar chains of human placental fibronectin: 1) nine sugar chains are included in one molecule; 2) all sialic acid residues are exclusively linked at the C-3 position of the galactose residues; 3) bi-, tri-, and tetraantennary complex-type oligosaccharides with the Man alpha 1----6(Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4 (+/- Fuc alpha 1----6)-GlcNac as their cores were found; 4) the bisecting N-acetylglucosamine residue and the Gal beta 1----4GlcNAc beta 1----repeating groups are included in some of the sugar chains.     

Structures of the sugar chains of rabbit immunoglobulin G: occurrence of asparagine-linked sugar chains in Fab fragment

The asparagine-linked sugar chains of rabbit immunoglobulin G (IgG) and its Fc and Fab fragments were quantitatively liberated from the polypeptide portions by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. After fractionation by paper electrophoresis, lectin chromatography, and gel filtration, their structures were studied by sequential exoglycosidase digestion in combination with methylation analysis. Rabbit IgG was shown to contain 2.3 mol of asparagine-linked sugar chains per molecule distributed in both the Fc and Fab fragments. The sugar chains were of the biantennary complex type containing four cores: Man alpha 1----6(Man alpha 1----3)(+/- GlcNAc beta 1----4)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)-GlcNAc. A total of 16 distinct neutral oligosaccharide structures was found after sialidase treatment. The galactose residue in the monogalactosylated oligosaccharides was present on either the alpha 1----3 or alpha 1----6 side of the trimannosyl core. The Fab fragments contained neutral, monosialylated, and disialylated oligosaccharides, whereas the Fc fragment contained only neutral and monosialylated structures. The oligosaccharides isolated from the Fab fragments also contained more galactose and bisecting N-acetylglucosamine residues than those from the Fc fragments.     

Mucin biosynthesis: characterization of rabbit small intestinal UDP-N-acetylglucosamine:galactose beta-3-N-acetylgalactosaminide (N-acetylglucosamine----N-acetylgalactosamine) beta-6-N-acetylglucosaminyltransferase

We have characterized a UDP-GlcNAc:Gal beta-3-GalNAc (GlcNAc----GalNAc) beta-6-N-acetylglucosaminyltransferase from rabbit small intestinal epithelium by using freezing point depression glycoprotein as the acceptor. Optimal enzyme activity was obtained at pH 7.0-7.5, at 3 mM MnCl2, and at 0.08% Triton X-100. Ca2+, Mg2+, and Ba2+ also enhanced enzyme activity. The apparent Michaelis constant was 4.80 mM for freezing point depression glycoprotein, 0.59 mM for periodate-treated porcine submaxillary mucin, 0.49 mM for Gal beta 1----3 GalNAc alpha Ph, and 1.03 mM for UDP-GlcNAc. No enzyme activity was observed when asialo ovine submaxillary mucin was used as the acceptor. The 14C-labeled oligosaccharide obtained by alkaline borohydride treatment of the product was shown to be a homogeneous trisaccharide by compositional analysis, Bio-Gel P-4 gel filtration, and high-performance liquid chromatography. The structure of the trisaccharide was identified as Gal beta 1----3-(GlcNAc beta 1----6)GalNAc-H2 by (a) identification of 2,3,4,6-tetramethyl-1,5-diacetylgalactitol and 1,4,5-trimethyl-3,6-diacetyl-2-N-methylacetamidogalactitol by gas-liquid chromatography-mass spectrometry and (b) the complete cleavage of the newly formed glycosidic bond by jack bean beta-hexosaminidase. The structure of the trisaccharide was confirmed by 1H nuclear magnetic resonance (270 MHz) and also by periodate oxidation of the trisaccharide followed by NaBH4 reduction, 4 N HCl hydrolysis, a second NaBH4 reduction, and the identification of threosaminitol on an amino acid analyzer. By acceptor competition studies, the enzyme activity was shown to be a much N-acetylglucosaminyltransferase. We postulate that this glycosyltransferase may play a key role in the regulation of mucin oligosaccharide synthesis.     

Regulation of glycosylation. The influence of protein structure on N-linked oligosaccharide processing

The Sindbis virus glycoproteins, E1 and E2, comprise a useful model system for evaluating the effects of local protein structure on the processing of N-linked oligosaccharides by Golgi enzymes. The conversion of oligomannose to N-acetyllactosamine (complex) oligosaccharides is hindered to different extents at the four glycosylation sites, so that the complex/oligomannose ratio decreases in the order E1-Asn139 greater than E2-Asn196 greater than E1-Asn245 greater than E2-Asn318. The processing steps most susceptible to interference were deduced from the oligosaccharide compositions at hindered sites in virus from baby hamster kidney cells (BHK), chick embryo fibroblasts (CEF), and normal and hamster sarcoma virus (HSV)-transformed hamster fibroblasts (Nil-8). Persistence of Man6-9GlcNAc2 was taken to indicate interference with alpha 2-mannosidase(s) I (alpha-mannosidase I), Man5GlcNAc2, with UDP-GlcNAc:alpha-D-mannoside beta 1----2-N-acetylglucosaminyltransferase I (GlcNAc transferase I), and unbisected hybrid glycans, with GlcNAc transferase I-dependent alpha 3(alpha 6)-mannosidase (alpha-mannosidase II). Taken together, the results indicate that all four sites acquire a precursor oligosaccharide with equally high efficiency, but alpha-mannosidase I, GlcNAc transferase I, and alpha-mannosidase II are all impeded at E2-Asn318 and, to a lesser extent, at E1-Asn245. In contrast, sialic acid and galactose transfer to hybrid glycans (in BHK cells) is virtually quantitative even at E2-Asn318. E2-Asn318 carried no complex oligosaccharides, but the structures of those at E1-Asn245 indicate almost complete GlcNAc transfer by UDP-GlcNAc:alpha-D-mannoside beta 1----2-N-acetylglucosaminyltransferase II (GlcNAc transferase II), galactosylation, and sialylation. Because the E2-Asn318 and E1-Asn245 glycans have previously been shown to be less accessible to a steric probe than those at E2-Asn196 or E1-Asn139, a simple explanation for these results would be that alpha-mannosidase I, GlcNAc transferase I, and alpha-mannosidase II are more susceptible to steric hindrance than are the later processing steps examined. Finally, in addition to these site-specific effects, the overall extent of viral oligosaccharide processing varied with host and cellular growth status. For example, alpha-mannosidase I processing is more complete in BHK cells compared to CEF, and in confluent Nil-8 cells compared to subconfluent or HSV-transformed Nil-8 cells.     

The structure of the asparagine-linked sugar chains of bovine brain ribonuclease

The asparagine-linked sugar chains of bovine brain ribonuclease were quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis. After N-acetylation, they were converted into radioactively-labeled oligosaccharides by NaB3H4 reduction. The radioactive oligosaccharide mixture was fractionated by ion-exchange chromatography, and the acidic oligosaccharides were converted into neutral oligosaccharides by sialidase digestion. The neutral oligosaccharides were then fractionated by Bio-Gel P-4 column chromatography. Structural studies of each oligosaccharide by sequential exoglycosidase digestion in combination with methylation analysis revealed that bovine brain ribonuclease showed extensive heterogeneity. It contains bi- and tri-antennary, complex-type oligosaccharides having alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-beta-D-Manp -(1----4)-beta-D- GlcpNAc-(1----4)-[alpha-L-Fucp-(1----6)]-D-GlcNAc as their common core. Four different outside oligosaccharide chains, i.e., beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----, alpha-Neu5Ac-(2----6)-beta-D- Galp-(1----4)-beta-D-GlcpNAc-(1----, alpha-Neu5Ac-(2----3)-beta-D-Galp-(1----4)- beta-D-GlcpNAc-(1----, and alpha-D-Galp-(1----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----, were found. The preferential distribution of the alpha-D-Galp-(1----3)-beta-D-Galp-(1----4)-beta-D-GlcpNAc group on the alpha-D-Manp-(1----6) arm is a characteristic feature of the sugar chains of this enzyme.     

Comparative study of the sugar chains of factor VIII purified from human plasma and from the culture media of recombinant baby hamster kidney cells.

The asparagine-linked sugar chains of blood coagulation factor VIII preparations purified from human plasma of blood group A donors and from the culture media of recombinant BHK cells were released as oligosaccharides by hydrazinolysis. These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis. Most of the acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives. The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial chromatography on immobilized lectin columns and Bio-Gel P-4 column. Structural study of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that both factor VIII preparations contain mainly high mannose-type and bi-, tri-, and tetra-antennary complex-type sugar chains. Some of the biantennary complex-type sugar chains from human plasma factor VIII contain blood group A and/or H determinant, while those from recombinant product do not. Some of the bi-, tri- and tetra-antennary complex-type sugar chains of the recombinant factor VIII contain the Gal alpha 1----3Gal group. A small number of the triantennary complex-type sugar chains from both preparations was found to contain the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----4 (Gal beta 1----4GlcNAc beta 1----2)Man group. Studies of pharmacokinetic parameters of the recombinant factor VIII infused into baboons revealed that its half-life in blood circulation is similar to that of plasma derived factor VIII, suggesting that the oligosaccharide structural differences between them do not affect the fate of factor VIII in vivo.     

Structural study of the asparagine-linked oligosaccharides of lipophorin in locusts

The complete structure of oligosaccharides from locust lipophorin was studied. The asparagine-linked oligosaccharides were first liberated from the protein moiety of lipophorin by digestion with almond glycopeptidase (N-oligosaccharide glycopeptidase, EC 3.5.1.52). Two major oligosaccharides (E and F), separated by subsequent thin-layer chromatography, were analyzed by methylation analysis and 1H-NMR. Based on the experimental data, the whole structure of oligosaccharide E was identified as Man alpha 1----2Man alpha 1----6(Man alpha 1----2Man alpha 1----3) Man alpha 1----6(Man alpha 1----2Man alpha 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4GlcNAc. The data also revealed that oligosaccharide F is identical with oligosaccharide E in the structure, except for one glucose residue that is linked to the nonreducing terminal Man alpha 1----2 residue.     

Structures of the asparagine-linked sugar chain of glucose transporter from human erythrocytes

The asparagine-linked sugar chain of glucose transporter from human erythrocytes was quantitatively released as oligosaccharides from the polypeptide backbone by hydrazinolysis. They were converted to radioactive oligosaccharides by NaB3H4 reduction after N-acetylation and fractionated by anion-exchange column chromatography and Bio-Gel P-4 column chromatography after sialidase treatment. Structural study of each oligosaccharide by exo- and endoglycosidase digestion and methylation analysis indicated that the glycoprotein contains a high-mannose-type oligosaccharide, Man9.GlcNAc.GlcNAc, and biantennary complex-type oligosaccharides with Man alpha 1----6(+/- GlcNAc beta 1----4)(Man alpha 1----3) Man beta beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc as their cores and the poly-N-acetyllactosamine composed of about 16 N-acetyllactosaminyl units as their outer chains. These structural features of the sugar moiety of glucose transporter are quite different from those of two major intrinsic glycoproteins of human erythrocytes, glycophorin A and band 3.     

Deglycosylation studies on tracheal mucin glycoproteins

Following several model experiments, conditions were developed for optimal deglycosylation of tracheal mucin glycoproteins. Exposure of rigorously dried material to trifluoromethanesulfonic acid at 0 degree C for up to 8 h results in cleavage of essentially all fucose, galactose, and N-acetylglucosamine, about 80% of the N-acetylneuraminic acid (NeuNAc), and a variable amount of N-acetylgalactosamine (GalNAc), the sugar involved in linkage to protein. Residual N-acetylneuraminic acid is sialidase susceptible and apparently in disaccharide units, presumably NeuNAc2----GalNAc. The remaining N-acetylgalactosamine is mostly present as monosaccharides, and a few Gal beta 1----3GalNAc alpha units are also present; both are cleaved by appropriate enzymatic treatment. The saccharide-free proteins obtained from either human or canine mucin glycoproteins have molecular weights of about 100,000 and require chaotropic agents or detergents for effective solubilization.